EBOLA ELISA

(Student CLASS COPY)

Objective:

You have been assigned by your boss at the County Health Department to analyze the serum of Crash survivors located in W. Africa. Some of the serum samples are from the survivors and some serum is from various animals thought to be a reservoir for Ebola (Bats, Monkeys, Elephants, etc etc.). You will use an ELISA to determine whether any of the samples contain antibodies against an epitope found on the protein coat of the EBOLA virus (called the H5 epitope). (You will each test your own samples.)

This protocol is designed to detect antibodies raised against the Ebola virus. Any individual that has been exposed to any virus or bacteria that cause disease (such as FLU, AIDS, Measles, Chicken Pox, Small Pox etc.) will have elevated antibody levels against these agents. This assay is designed to look for those elevated antibody levels

Pre-lab Questions:

  1. What is an antigen?
  2. What is an antibody?
  3. How do antigens and antibodies interact?
  4. What does ELISA stand for?
  5. Why do you need to assay positive and negative control samples as well as your experimental samples?
  6. Why did the health department want you to test the dead animals as well as your antiserum?

Materials: (1 group)

  • 96-well plate
/
  • 650 l peroxidase substrate (blue tube)

  • 20 ml Wash Buffer
/
  • paper towels

  • 750 l H5 Ebola antigen (green tube)
/
  • watch or clock with second hand

  • 175l + control human serum (pink tube)
/
  • safety glasses

  • 175l – control human serum (purple tube)
/
  • 1-P-200 pipet

  • 175l Your serum (orange tube)
/
  • 1 box of tips (20-200 l)

  • 175l dead animal serum (clear tube)
/
  • 1 transfer pipet

  • 525 l rabbit anti-animal IgG-peroxidase (brown tube)

  • 175l rabbit anti-human IgG-peroxidase (yellow tube)

  • 1 permanent marker

Protocol:

  1. You will run your samples in triplicate (three wells for each sample). In the 96-well template below record the location of your samples.
  1. Add 50 l of H5 Antigen (green tube) to each of the 12 wells. Discard the pipet tip.
  1. Incubate 5 minutes at room temperature. This allows the antigen to bind to the plastic wells.
  1. Turn the 96-well plate upside down on a stack of paper towels and bang it forcefully several times to remove any unbound antigen. Discard the top layers of paper towels.
  1. Use a transfer pipet to fill each well with wash buffer. Be careful not to touch the sides of the wells with the tip. PLEASE REUSE PIPET FOR FUTURE WASHES. Remove buffer as described in step #4.
  1. Add 50 l of each serum to the wells you designated above on your 96-well template. Be sure to use a fresh pipet tip for each serum.
  • 3 wells (+) control (pink tube)
  • 3 wells ( - ) control (purple tube)
  • 3 wells human (orange tube)
  • 3 wells dead animal (clear tube)
  1. Incubate 5 minutes at room temperature. This allows anti-H5 antibodies, if present, to bind to H5 antigen.
  1. Remove serum as described in step #4.
  1. Use a transfer pipet to fill each well with wash buffer. Remove buffer as described in step #4.
*If stopping at this point:

Refill all wells halfway with wash buffer and cover with a lid if provided. If there is no lid, CAREFULLY slide your plate into a labeled Ziploc™bag, and give it to your teacher to store in the refrigerator until your next class meeting.

  1. Add 50 l of rabbit anti-animal IgG-peroxidase (brown tube) to the wells that received animal serum. Discard the pipet tip.
  • 3 wells (+) control
  • 3 wells ( - ) control
  • 3 wells dead animal
  1. Using a new pipet tip, add 50 l of rabbit anti-human IgG-peroxidase (yellow tube) to the wells that received human serum. Discard the pipet tip.
  • 3 wells human
  1. Incubate for 5 minutes at room temperature.
  1. Remove the rabbit antibodies from the wells as described in step #4.
  1. Wash each well three times with wash buffer as described in step #9.
  1. Add 50 l of peroxidase substrate (blue tube) to each well.Be careful not to touch the sides of the wells with the tip.
  1. Incubate 5 minutes and then record your results.

Analysis:

1. Did your sample contain the antigen? How do you know?

2. Why did you add a primary antibody?

3. Why did you add a secondary antibody?

4. Why did you assay your samples in triplicate?

5. What antibody-based tests can you buy at your local pharmacy?

Conclusion:

As an officer of the county health department, compose a letter to the survivors that summarizes the results of your ELISA and includes your recommendations for investigating the next step in this potential outbreak.

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